The coccidian parasite Toxoplasma gondii is a serious pathogen of humans and livestock worldwide. T. gondii is an obligate intracellular parasite that causes abnormal fetal neurological development and encephalitis in immunocompromised patients. The drugs available against T. gondii are toxic, must be taken in combinations, and are not capable of eliminating a chronic infection. Our goal is to identify and functionally characterize T. gondii genes that are essential for the establishment of a chronic infection in mice, but not necessary for cell culture growth. We are terming these virulence genes. For this goal, we have adapted signature-tagged mutagenesis (STM) for T. gondii. STM was originally used to mutagenize Salmonella with a unique sequence tag. Virulence genes were isolated by comparing the tags represented in the inoculum versus the bacteria recovered from an infected host. A library of 4900 T. gondii STM clones has been screened for their ability to establish a chronic infection in mice using oral inoculation of in vitro derived cysts. Of these 4900 mutants, 4500 have derived their mutation from chemical mutagenesis and 400 were generated by insertional mutagenesis. We have uncovered 180 potential mutants defective in their ability to cause a chronic infection in mice. Of the 180 STM strains, seven are insertional mutants. When retested in mice, four of the insertional mutants were at least 100-fold less virulent (as measured by LD50). The genes flanking the insertion site for these mutants have been isolated and are currently being analyzed. Antiserum generated against one of the predicted proteins recognizes a 120 KD protein only during stress conditions. Another gene predicts a protein with a signal sequence and several transmembrane domains. We will then use a combination of molecular genetics, biochemistry, and cell biological approaches to characterize the function(s) of these genes. The large numbers of potential mutants imply that many genes are involved in virulence; these will be most easily isolated by expanding and screening the library of insertional mutants. We will generate and analyze 6000 new insertional mutants. It will be important to investigate if parallel virulence genes exist in other pathogenic coccidians that are not as easy to manipulate in vitro, but that have genome sequencing projects such as Plasmodium, and Cryptosporidium. [unreadable] [unreadable]